JMJD5 cleaves monomethylated histone H3 N-tail under DNA damaging stress.

The histone H3 N-terminal protein domain (N-tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N-tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N-tail at the carboxyl side of monomethyl-lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl-lysine (Kme2), trimethyl-lysine (Kme3), or unmethyl-lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N-tail cleavage in gene expression regulation.

Three new alternative splicing variants of human cytochrome P450 2D6 mRNA in human extratumoral liver tissue.

AIM: To identify the new alternative splicing variants of human CYP2D6 in human extratumoral liver tissue with RT-PCR and sequencing. METHODS: Full length of human CYP2D6 cDNAs was amplificated by reverse transcription-polymerase chain reaction (RT-PCR) from a human extratumoral liver tissue and cloned into pGEM-T vector. The cDNA was sequenced. Exons from 1 to 4 of human CYP2D6 cDNAs were also amplificated by RT-PCR from extratumoral liver tissues of 17 human hepatocellular carcinomas. Some RT-PCR products were sequenced. Exons 1 to 4 of CYP2D6 gene were amplified by PCR from extratumoral liver tissue DNA. Two PCR products from extratumoral liver tissues expressing skipped mRNA were partially sequenced. RESULTS: One of the CYP2D6 cDNAs had 470 nucleotides from 79 to 548 (3' portion of exons 1 to 5' portion of exon 4), and was skipped. Exons 1 to 4 of CYP2D6 cDNA were assayed with RT-PCR in 17 extratumoral liver tissues. Both wild type and skipped mRNAs were expressed in 4 samples, only wild type mRNA was expressed in 5 samples, and only skipped mRNA was expressed in 8 samples. Two more variants were identified by sequencing the RT-PCR products of exons 1 to 4 of CYP2D6 cDNA. The second variant skipped 411 nucleotides from 175 to 585. This variant was identified in 4 different liver tissues by sequencing the RT-PCR products. We sequenced partially 2 of the PCR products amplified of CYP2D6 exon 1 to exon 4 from extratumoral liver tissue genomic DNA that only expressed skipped mRNA by RT-PCR. No point mutations around exon 1, intron 1, and exon 4, and no deletion in CYP2D6 gene were detected. The third variant was the skipped exon 3 , and 153 bp was lost. CONCLUSION: Three new alternative splicing variants of CYP2D6 mRNA have been identified. They may not be caused by gene mutation and may lose CYP2D6 activity and act as a down-regulator of CYP2D6.

Stable expression of human cytochrome P450 2D6*10 in HepG2 cells.

AIM: Over 90% of drugs are metabolized by the cytochrome P-450 (CYP) family of liver isoenzymes. The most important enzymes are CYP1A2, 3A4, 2C9/19, 2D6 and 2E1. Although CYP2D6 accounts for <2% of the total CYP liver enzyme content, it mediates metabolism in almost 25% of drugs. In order to study its enzymatic activity for drug metabolism, its cDNA was cloned and a HepG2 cell line stably expressing CYP2D6 was established. METHODS: Human CYP2D6 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from human liver tissue and cloned into pGEM-T vector. cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A cell line was established by transfecting the recombinant plasmid of pREP9-CYP2D6 to hepatoma HepG2 cells. Expression of mRNA was validated by RT-PCR. Enzyme activity of catalyzing dextromethorphan O-demethylation in postmitochondrial supernatant (S9) fraction of the cells was determined by high performance liquid chromatography (HPLC). RESULTS: The cloned cDNA had 4 base differences, e.g. 100 C-T, 336 T-C, 408 C-G and 1 457 G-C, which resulted in P34S, and S486T amino acid substitutions, and two samesense mutations were 112 F and 136 V compared with that reported by Kimura et al (GenBank accession number: M33388). P34S and S486T amino acid substitutions were the characteristics of CYP2D6*10 allele. The relative activity of S9 fraction of HepG2-CYP2D6*10 metabolized detromethorphan O-demethylation was found to be 2.31 +/- 0.19 nmol/min(-1)/mg(-1) S9 protein (n=3), but was undetectable in parental HepG2 cells. CONCLUSION: cDNA of human CYP2D6*10 can be successfully cloned. A cell line, HepG2-CYP2D6*10, expressing CYP2D6*10 mRNA and having metabolic activity, has been established.

Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line.

AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many small-molecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens, procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1. METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA) cytotoxicity and micronucleus test. RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al (GenBank access No. J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162 +/- 0.025 nmol.min(-1).mg(-1) S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells. CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity, mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.

N-methyl-N'-nitro-N-nitrosoguanidine sensitivity, mutator phenotype and sequence specificity of spontaneous mutagenesis in FEN-1-deficient cells.

Intact pZ189 DNA was allowed to replicate in FL-FEN-1(-) cell line that was established in this laboratory in which the expression of FEN-1 gene was blocked by dexamethasone-inducible expression of antisense RNA to FEN-1. E. coli MBM7070 was transfected with the replicated plasmid, and those with mutations in the supF gene were identified. The frequency of mutants that did not contain recognizable changes in the electrophoretic mobility of the plasmid DNA was scored. The frequency of such mutants was 19.1 x 10(-4) (34/17781), significantly higher than those of 2.9 x 10(-4) (4/13668) and 3.0 x 10(-4) (3/9857) in the corresponding controls, respectively. Sequence analysis of the supF genes of these mutants showed that all (37/37) the base substitutions occurred at C:G base pairs; 68% (23/37) of the base substitutions were base transversions, while 32% (12/37) were transitions. Approximately 76% (23/37) of these base substitutions occurred frequently at nine positions; two of these sites contain triple pyrimidine (T or C) repeat upstream to the mutated base; four of these sites consist of 5'-TTN1N2 and mutations occurred at N1 site sequence; another two sites have the characteristics of triple A flanked at both 5' and 3' side by TCT, with the base substitution occurring at C in the context sequence. These data suggested that these sites are the hot spot of mutagenesis in plasmid replicated in FEN-1-deficient cells. Besides the mutator phenotype of the FEN-1-deficient cell, it was also demonstrated that FEN-1-deficient cell exhibited an increased N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) sensitive phenotype.

Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing.

AIM: The human cytochrome P-450 2C18(CYP2C18) has been characterized. However, the protein has not been purified from liver and very little is known regarding the specific substrate of CYP2C18. In order to study its enzymatic activity for drug metabolism, the CYP2C18 cDNA was cloned and a stable CHL cell line expressing recombinant CYP2C18 was established. METHODS: The human CYP2C18 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR) from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2C18 to Chinese hamster lung (CHL) cell. The enzyme activity of CYP2C18 catalyzing oxidation of tolbutamide to hydroxytolbutamide in postmitochondrial supernant(S9) fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cloned cDNA segment was identical to that of reported by Romkes et al (GenBank accession number: M61856, J05326). The S9 fraction of the established cell line metabolizes tolbutamide to hydroxytolbutamide. Tolbutamide hydroxylase activity was found to be 0.509+/-0.052 micromol x min(-1) x g(-1) S9 protein or 8.82+/-0.90 mol x min(-1) x mol(-1) CYP, but was undetectable in parental CHL cell. In addition, we have identified a CYP2C18 cDNA clone with exon 5 missing. CONCLUSION: The cDNA of human CYP2C18 was successfully cloned and a cell line, CHL-CYP2C18, efficiently expressing the protein of CYP2C18, was established. A spliced variant of CYP2C18 with exon 5 missing was identified in the cloning process.

Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells.

AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T>C, 1146C>T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.

Response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and its role in mutagenesis.

AIM: To understand the response of human REV3 gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and its role in human mutagenesis. METHODS: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRT locus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FL-REV3(-) by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3(-) and FL-REV3(-). The blockage effect of REV3 was measured by combination of reverse transcription-polymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3 protein level. RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27 fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P<0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P<0.05). In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein. MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P<0.05) and 5.8 fold (P<0.01) respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3 was blocked by antisense RNA, and almost recovered to their spontaneous mutation levels. The spontaneous HPRT mutation was disappeared in REV3-disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66 x 10(-6) in FL cells to 0.14 x 10(-6) in transgenic cells as well (P<0.01). CONCLUSION: The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.

ATM and ATR: sensing DNA damage.

Cellular response to genotoxic stress is a very complex process, and it usually starts with the "sensing" or "detection" of the DNA damage, followed by a series of events that include signal transduction and activation of transcription factors. The activated transcription factors induce expressions of many genes which are involved in cellular functions such as DNA repair, cell cycle arrest, and cell death. There have been extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process, including the mitogen-activated protein kinases (MAPKs) cascade. Although the initial activation of protein kinase cascade is not fully understood, human protein kinases ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) are emerging as potential sensors of DNA damage. Current progresses in ATM/ATR research and related signaling pathways are discussed in this review, in an effort to facilitate a better understanding of genotoxic stress response.

Establishment of Cell Line with the FEN-1 Gene Blocked by Its Antisense.

FEN-1 is a structure-specific endo/exonuclease, which is involved in the process of both DNA duplication and DNA repair. In this work a mammalian expression vector expressing antisense FEN-1 gene fragment pMAMneoAmp(-)FNB(-) was constructed, after cloning the NcoI-BamHI fragment of FEN-1 gene into the mammalian expression vector pMAMneoAmp(-) in antisense orientation. After FL cell was transfected with pMAMneoAmp(-)FNB(-) and selected by G418, the FL-FEN-1(-) cell line, in which the FEN-1 gene expression was blocked, was established. It was found that the growth of FL-FEN-1(-) was decreased upon the induction with dexamethasone and its T(D) was 3.03 d, while the T(D) of controls FL and FL-M induced with dexamethasone was 2.03 and 2.22 d, respectively, and the T(D) of the FL-FEN-1(-) cell without dexamethasone was 2.38 d.

The Influence of FEN-1 Gene on Cell Cycle and Genetic Stability.

FEN-1 is essential in the cell replication, repair and in the maintenance of cellular genetic stability. In this report, it was verfied that FEN-1 antisense mRNA fragment was expressed in the cell line FL-FEN-1(-),constructed in our lab, blocking FEN-1 gene expression. It was found by the flow cytometer analysis that the cell cycle of FL-FEN-1(-) cells was delayed in the S-phase DNA synthesis process and arrested in G(1) phase. In a mutation assay, based on the shuttle-plasmid pZ189, the spontaneous mutation frequency of SupF tRNA gene in the plasmid in the FL-FEN-1(-) cells was 19.1x10(4),while it was 2.9x10(4) and 3.0x10(4) in the control cells FL and FL-M, respectively. Further study showed that nontargeted mutation frequency of the FL-FEN-1(-) cell induced by MNNG was almost the same as the control, indicating that the mutants derived from the block of FEN-1 gene and the nontargeted mutants may be formed through different passways. The FL-FEN-1(-) cells exhibit increased sensitivity to alkylating agent MNNG.

[Establishment of a cell line with antisense-blocked POLH and the role of POLH in alkylating agent MNNG induced nontargeted mutagenesis].

OBJECTIVE: To investigate the function of POLH(polymerase eta) through establishment of the POLH gene-blocked cell line FL-POLH(-). METHODS: A mammalian expression vector expressing antisense POLH gene fragment pMAMneo-amp-POLHA (-) was constructed by cloning the 1473 - 2131 fragment of POLH gene into the mammalian expression vector pMAMneo-amp(-) in antisense orientation. The FL cells were transfected with this antisense RNA expressing vector and selected by G418. The mutation assay was conducted using the shuttle plasmid pZ189. RESULT: The spontaneous mutation frequency of SupF tRNA gene in the plasmid replicated in the FL-POLH(-) was 13.5 x 10(-4), while it was 4.9x10(-4) and 3.7x10(-4) in the control cells FL and FL-M, respectively. The nontargeted mutation frequency of SupF tRNA gene decreased in the plasmid replicated in these cell lines pretreated with MNNG. CONCLUSION: POLH plays an important role in maintenance of genetic stability and genesis of nontargeted mutation.

[Establishment of a HepG2 cell line stably expressing human cytochrome P450 1A2 and its metabolic activity].

OBJECTIVE: To establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metabolic activity. METHODS: The human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT PCR. The metabolic activation of HepG2 CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test. RESULT: The HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild type HepG2 cells. CONCLUSION: The established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.

[Proteomic analysis of cellular responses to low concentration of N-methyl-N'-nitro-N-nitrosoguanidine in human amnion FL cells].

OBJECTIVE: To investigate the protein profile after treatment of low concentration of N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) in human FL cells. METHODS: After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images were analyzed with 2D analysis software. The differentially expressed protein spots were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULT: More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). There were 18 protein spots detected only after MNNG treatment, while 13 protein spots were detected only in the control cells. Moreover, the levels of another 31 proteins were either increased or decreased in MNNG-treated FL cells. And some of the proteins were identified by MALDI-TOF-MS. CONCLUSION: There are significant alterations of protein profile after MNNG attack.

[Altered of zinc finger proteins expression in FL cells following benzo [a] pyrene treatment].

OBJECTIVE: To understand benzo[a]pyrene (B[a]P) mediated cellular responses, and to provide clues to explore molecular mechanism of mutagenesis and carcinogenesis induced by B[a]P. METHODS: Two-dimensional electrophoresis (2-DE) was used to investigate the protein expression levels of FL cells after B[a]P exposure, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) combined with database search was applied to identify the differentially expressed proteins. RESULT: Statistical analysis showed that the volumes of 47 protein spots were altered after B[a]P treatment (P<0.05) and 23 proteins were successfully identified, including zinc finger proteins, SWI/SNF related protein, Bromo domain containing domain and other proteins. CONCLUSION: These affected proteins may be involved in the cellular responses to B[a]P exposure, and may mediate the B[a]P induced mutagenesis and carcinogenesis.

[Activation of transcription factors induced by low concentration of N-methyl-N'-nitro-N-nitrosoguanidine].

OBJECTIVE: To investigate the effect of MNNG on some of the transcription factors such as NF- kappaB, CREB, AP-1 and c-Myc. METHODS: The activities of these transcription factors were measured by transient transfection assay of SEAP vectors. RESULT: The expressions of AP-1, CREB and NF- kappaB driven reporter genes were elevated for about 1.3, 1.4 and 1.3 times in MNNG-treated cells, respectively, as compared to untreated controls. However, the exposure of MNNG had no effect on the activity of c-Myc. CONCLUSION: The activation of certain transcription factors might be involved in the process of untargeted mutation induced by low concentration of MNNG treatment.

[Cloning and bioinformatics of human REV3 gene promoter region and its response to carcinogen N-methyl-N'-nitro-N-nitrosoguanidine].

OBJECTIVE: To understand the up regulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). METHODS: Bioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites. Cloning of human REV3 gene promoter region was performed by nested PCR. Response of human REV3 gene promoter to the chemical carcinogen MNNG was measured by transient transfection assay based on the dual luciferase reporter assay system. RESULT: Bioinformatic analysis showed that human REV3 gene promoter region was located on chromosome 6 PAC clone RP3-415N12, and that the hypothetical promoter region contained promoter sequences, rich CpG islands, and putative recognition sites for several transcriptional factors, including AP-1/c-Jun/c-Fos, AP-2, STAT, CREBP, and NF-kappaB. Reconstructed reporter plasmid pGL3- 2582 was established by inserting 2582 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. Transient transfection assay showed the hypothetical REV3 promoter region had promoter function, and it responded to MNNG treatment (P<0.01). CONCLUSION: Human mutator REV3 gene promoter region has been successfully cloned. The response of REV3 promoter region to MNNG suggests that REV3 gene can be regulated at transcriptional level under conditions of genotoxic stress.

[Activation of nucleus-independent signals triggered by N-methyl-N'-nitro-N- nitrosoguanidine].

OBJECTIVE: To study the effect of MNNG on inducement of non-targeted mutation and activation of several cellular signal transduction pathways, and to determine whether the activation of these signaling pathways was dependent on the DNA-damage. METHODS: Vero cells were enucleated by discontinuous density centrifugation. The PKA activities were measured by enzyme-linked immunosorbent assay. The status of cell membrane receptors was studied with immunofluorescent staining and confocal microscopy. RESULT: In enucleated cytoplasts, MNNG-treatment increased PKA activity for about 2.3-fold in accordance with the 2.7-fold up-regulation of PKA activity in whole vero cells exposed to MNNG. The clustering of cell surface receptors of epidermal growth factor and tumor necrosis factor alpha was also observed in cells exposed to MNNG; this phenomenon was also found in enucleated cells. CONCLUSION: The results indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damage.

[Stereoselective determination of propranolol enantiomer in transgenic cell lines expressing human cytochrome P450].

OBJECTIVE: To establish a chiro chromatography for studying the stereoselective metabolism of propranolol (PL) in S(9) incubates prepared from transgenic cell lines expressing human cytochrome P450. METHODS: The concentration of each enantiomer in S(9) incubates was determined through precolumn derivatization with GITC, followed by RP-HPLC assay using S-(+)-propafenone as internal standard. RESULTS: Baseline separations among the diastereomers of S(-)-P, internal standard and R(+)-PL were achieved on Shimpack CLC C(18)ODS column, with UV detection and methanol:water:glacial acetic acid (67/33/0.05,v/v/v) as mobile phase. The assay was simple, accurate, precise and specific. The linear range was from 5 to 500 micromol/L for each enantiomer. The limit of quantitation (LOQ) for the method was 5 micromol/L for the S(-)-and R(+)-PL, respectively (n=5, RSD<10%). The analytical method afforded average recoveries of 98.7 and 98.1% for S(-)- and R(+)-PL, respectively. The reproducibility of the assay was good (RSD<10%). The time-dependent studies showed that PL had the stereoselectivity of S-(-)-isomer in metabolism via CYP2C18 and the stereoselectivity of R-(+)-isomer in metabolism via CYP2C9. CONCLUSION: The method allows to study of stereoselective metabolism of PL in vitro.